It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, attempts have been made to moderate the production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to achieve therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate expression of genes which are responsible for the formation of undesired protein.
One method for inhibiting specific gene expression is by the use of oligonucleotides or modified oligonucleotides as "antisense" agents. As so used, oligonucleotides or modified oligonucleotides are selected to be complimentary to a specific, target, messenger RNA (mRNA) sequence. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides or modified oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted.
The use of oligonucleotides, modified oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic and diagnostic use is actively being pursued by many commercial and academic groups. While the initial suggested mode of activity of antisense agents was via hybridization arrest, several additional mechanisms or terminating antisense events have also been studied in relation to antisense use in therapeutics. In addition to hybridization arrest these include cleavage of hybridized RNA by the cellular enzyme ribonuclease H (RNase H), RNA catalytic or chemical cleaving and cross-linking. Various reviews in the scientific literature summarize these studies. See, for example, Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (Cohen ed., 1989); Cook, P. D. Anti-Cancer Drug Design 1991, 6,585; Cook, P. D. Medicinal Chemistry strategies for Antisense Research, in Antisense Research & Applications, Crooke, et al., CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al., A. Chem. Rev. 1990, 90, 543; Walder, et al., Proc. Natl. Acad. Sci., USA, 1988, 85, 5011; and Dagle, et al., Antisense Research & Development, 1991, 1, 11.
Hybridization arrest denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides, such as those of Miller, et al., Anti-Cancer Drug Design, 1987, 2, 117-128, and .alpha.-anomer oligonucleotides are two extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
In the RNase H terminating event, activation of RNase H by a heteroduplex formed between a DNA type oligonucleotide or oligonucleotide analog and the targeted RNA results in cleavage of target RNA by the enzyme, thus destroying the normal function of the RNA. To date, the RNAse H enzyme has been found to be activated only by either natural phosphodiester DNA oligonucleotides or phosphorothioate DNA oligonucleotides. Walder, supra and Stein, et al., Nucleic Acids Research, 1988, 16, 3209-3221 describe the role that RNase H plays in the antisense approach.
oligonucleotides or modified oligonucleotides acting as chemical or catalytic RNA cleavers require either the attachment of pendent groups with acid/base properties to oligonucleotides or the use of ribozymes, i.e. RNAs having inherent catalytic properties. In the pendent group approach, the pendent group is not involved with the specific Watson-Crick hybridization of the oligonucleotide or oligonucleotide analog with the mRNA but is carried along by the oligonucleotide or oligonucleotide analog to serve as a reactive or non-reactive functionality. The pendent group is intended to interact with the mRNA in some manner to more effectively inhibit translation of the mRNA into protein. Such pendent groups have also been attached to molecules targeted to either single or double stranded DNA. Such pendent groups include intercalating agents, cross-linkers, alkylating agents, or coordination complexes containing a metal ion with associated ligands.
Cross-linking of a nucleic acid with a complimentary oligonucleotide or modified oligonucleotide is used to modulate RNA activity by disrupting the function of nucleic acids. To date this has primarily been achieved by cleaving the target. The known approaches using cross-linking agents, as well as alkylating agents and radical generating species, as pendent groups on oligonucleotides for antisense diagnostics and therapeutics have had several significant shortcomings. It is known to cross-link nucleic acids by exposure to UV light; however, such cross-linking is positionally uncontrollable. To overcome this lack of specificity, some workers have covalently cross-linked complementary strands of oligonucleotides at a specific site utilizing controlled chemistry. These workers attached a nitrogen mustard to either the 3' terminal ribose unit of an oligonucleotide or oligonucleotide analog via an acetal linkage or to the 5' end of an oligonucleotide or oligonucleotide analog via a phosphoramide linkage. On hybridization, the reactive mustards covalently cross-linked to the complementary strand via alkylation of the ternary heteroaromatic nitrogen atom at the 7-position of guanine or adenine: see Grineva et al., FEBS., 1973, 32, 351-355. Other workers have attached an .alpha.-bromomethylketone to the 4-position of a cytidine nucleotide which spans the major groove and alkylates the 7-position of a complementary guanine residue in a targeted strand: see Summerton et al., J. Mol. Bio., 1978, 122, 145-162; J. Theor. Biology, 1979, 78, 61-75; and U.S. Pat. No. 4,123,610. The alkylated bases formed under these conditions are quaternary charged species that are subject to rapid chemical degradation via imidazole ring opening followed by cleavage of the targeted strand. Meyer et. al., J. Am. Chem. Soc., 1989, 111, 8517, described attaching an iodoacetamidopropyl moiety to the 5-position of a thymidine nucleotide of DNA that alkylated the 7-position of a guanine nucleotide at a position two base pairs down the complementary strand.
Cross-linking may also be achieved by hybridization. For example, an N6,N6-ethano-adenine or N4,N4-ethanocytosine alkylates an appropriately positioned nucleophile in a complementary strand. This process has been designed to inactivate the normal function of the targeted DNA either by forming a stable adduct or by hydrolytic or enzymatic cleavage: see Mateucci et al., Nucleic Acids Res., 1986, 14, 7661; Tetrahedron Letters, 1987, 28, 2469-2472; Ferentz et al., J. Am. Chem. Soc., 1991, 113, 4000.
A serious deficiency of the early studies using oligonucleotides as antisense agents, particularly in the filed of therapeutics, resided in the use of unmodified oligonucleotides for these purposes. Such unmodified oligonucleotides are degraded by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes, hereinafter referred to as "nucleases."
To enhance the above mechanisms of action of antisense agents, a number of chemical modifications have been introduced into antisense agents, i.e. oligonucleotides and oligonucleotide analogs, to increase their therapeutic activity. Such modifications are designed to modify one or more properties including cellular penetration of the antisense agents, stabilization of the antisense agents to nucleases and other enzymes that degrade or interfere with their structure or activity in the body, enhancement of the antisense agents' binding to targeted RNA, modification of the mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, or improvement of the antisense agents' pharmacokinetic and pharmacodynamic properties.
To increase the potency of an oligonucleotide antisense agent by increasing its resistance to nucleases, modifications are most often introduced at the sugar-phosphate backbone, particularly on the phosphorus atom. Phosphorothioates, methyl phosphonates, phosphoramidites, and phosphotriesters have been reported to have various levels of resistance to nucleases. Other backbone modifications are disclosed as set forth in U.S. entitled "Backbone Modified Oligonucleotide Analogs," and "Heteroatomic Oligonucleotide Linkages," the disclosures of which are incorporated herein by reference to disclose more fully such modifications.
The phosphorothioate modified oligodeoxynucleotides are of particular use since, as noted above, they are capable of cleaving RNA by activation of RNase H upon hybridization to RNA. Hybridization arrest of RNA function may, however, play some part in their activity.
Other modifications to "wild type" oligonucleotides include functionalizing the nucleoside's naturally occurring sugar. Certain sugar modifications are disclosed as set forth, assigned to a common assignee hereof, entitled "Compositions and Methods for Detecting and Modulating RNA Activity and Gene Expression," the disclosure of which is incorporated herein by reference to disclose more fully such modifications. Heteroduplexes formed between RNA and oligonucleotides bearing 2'-sugar modifications, e.g. RNA mimics such as 2'-fluoro and 2'-alkoxy, do not support RNase H-mediated cleavage. These modified heteroduplexes assume an A form helical geometry as does RNA-RNA heteroduplexes, which also do not support RNase H cleavage. See Kawasaki, et al., J. Med. Chem., in press 1993; Lesnik, et al., Biochemistry, submitted 1993; Inoue, et al., Nucleic Acids Res. 1987, 15, 6131.
Further modifications include modification to the heterocyclic portions, rather than to the sugar-phosphate portions, of the nucleosides of an oligonucleotide or oligonucleotide analogue. Substitutions at the N-2, N-6 and C-8 positions of certain purines, such as hypoxanthine, guanine, or adenine have been reported: see, e.g., Harris et al., J. Am. Chem. Soc., 1991, 113, 4328-4329 (displacement of a 2-halogen or a 6-halogen group on a purine within an oligonucleotide to give a modified oligonucleotide wherein the purine ring of one of the nucleotides of the oligonucleotide is substituted at the 2-position or 6-position with phenylgylcinol); Johnson et al., J. Am. Chem. Soc., 1992, 114, 4923-4924 (post synthetic introduction of an 8-fluorenylamino group at the 2-position of deoxyguanosine residues in oligonucleotides via a new protecting group); Lee et al., Tetrahedron Letters, 1990, 31, 6773-6776 (introducing a pyrene molecule at the N-2 and N-6 position of purine nucleotides of an oligonucleotide as a model for derivatization with polycyclic aromatic hydrocarbons); Casale et al., J. Am. Chem. Soc., 1990, 112, 5264-5271 (introducing a 9-methyl anthracene group at the N-2 position of a 2'-deoxyguanosine nucleotide of an oligonucleotide causing destabilization of the DNA duplex); Kim et al., J. Am. Chem. Soc., 1992, 114, 5480-5481 (introducing a 6-fluoro purine nucleotide into an oligonucleotide followed by displacement of the fluorine with a polycyclic aromatic amine); Gao et al., J. Organ. Chem., 1992, 57, 6954-6959 (introducing a pentafluorophenyl group, i.e. --OC.sub.6 F.sub.S, at the N-6 position of deoxyguanosine for post-synthetic modification to 2-aminoadenosine or 6-O-methylguanosine); Gmeiner et al., Bioorg. Med. Chem. Letters, 1991, 1, 487-490 (formation of 6-N-(2-cholesteroloxycarboaminethyl) or 6-N-[2-(9-fluorenylcarboamino)ethyl] adenosine moieties via a 6-chloropurine riboside followed by introduction of these derivatized adenosine nucleosides into oligonucleotides).
The common way of functionalizing the C-8 position of purines is from 8-bromoadenosine derivatives. Roduit, et. al., |Nucleosides, Nucleotides, 1987, 6, 349, displaced an 8-bromo substituent with an N-protected cysteamine derivative to obtain an aminolinker. In a like manner a 2'-deoxyadenosine nucleotide containing an aminolinker at C-8 having fluorescein attached has been incorporated into an oligonucleotide. Biotin was coupled via a disulfide bridge and 4,5',8-trimethylpsoralen was coupled via sulfur and a 5 carbon atom long alkyl linker, also at C-8. See Pieles, et. al., Nucleic Acids Res., 1989, 17, 8967. In a like manner a dansyl group has been introduced at the C-8 via displacement of the 8-bromo group with a diamino linker and coupling of the dansyl functionality to the terminal amine. See Singh, et. al., Nucleic Acids Res., 1990, 18, 3339.
There still remains a great need for antisense agents that are capable of improved specificity and effectiveness both in binding and modulating mRNA or inactivating mRNA without imposing undesirable side effects. Further, heretofore, there has be no suggestion in the art of cross-linkages or methods of cross-linking that do not destroy the strands, that allow suitable conformations, that are useful on various sequences and at various positions within the sequences, or that allow normal ranges of features such as the "tilt" and "propeller twist" features found in naturally occurring nucleic acid duplexes. Accordingly, there also remains a long-felt need for nucleic acid cross-linkers and methods of cross-linking nucleic acids. The present invention addresses these as well as other needs by presenting novel oligonucleotide intermediates based on the core structure of the purine ring system.